Implantable devices and methods for evaluation of active agents

ABSTRACT

Devices for the local delivery of microdose amounts of one or more active agents, alone or in combination, in one or more dosages, to selected tissue of a patient are described. The devices generally include multiple microwells arranged on or within a support structure and contain one or more active agents, alone or in combination, in one or more dosages and/or release pharmacokinetics. In an exemplary embodiment, the device has a cylindrical shape, having symmetrical wells on the outside of the device, each well containing one or more drugs, at one or more concentrations, sized to permit placement using a catheter, cannula, or stylet. Optionally, the device has a guidewire, and fiber optics, sensors and/or interactive features such as remote accessibility to provide for in situ retrieval of information and modification of device release properties. In a preferred embodiment, the fiber optics and/or sensors are individually accessible to discrete wells.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.13/729,738 entitled “Implantable Devices and Methods for the Evaluationof Active Agents” by Robert I. Tepper, Jason Fuller, Oliver Jonas, andJohn Santini, filed on Dec. 28, 2012, which claims the benefit of andpriority to U.S. Provisional Application No. 61/582,009 entitled“Implantable Devices and Methods for the Evaluation of Active Agents” byRobert I. Tepper, Jason Fuller, Oliver Jonas, and John Santini, filed onDec. 30, 2011, and where permissible is incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The invention is generally related to devices, methods, systems, andkits for the evaluation of therapeutic agents in situ within tissues tobe treated in patients.

BACKGROUND OF THE INVENTION

In recent years, research has demonstrated that the progression of manydiseases is governed by molecular and genetic factors which are patientspecific. For example, it is now understood that cancer is driven bydiverse genetic and epigenetic factors which are often patient specific.As a result, disease progression and anti-cancer drug response is uniqueto every patient. In spite of this understanding, most clinicaltreatments still follow established standard-of-care guidelines andparadigms which fail to account for patient-specific factors.

Personalizing therapeutic treatments in view of the patient-specificmolecular and genetic factors offers the opportunity to improvetherapeutic outcomes. In order to tailor treatments in a patientspecific fashion, tools and methods of predicting and/or rapidlydetermining the response of a patient to particular drug regimens areneeded.

Therefore, it is an object of the invention to provide devices that canbe used to locally deliver discrete microdose quantities of one or moreactive agents to tissues in a patient, and which can be easily removedwith tissue remaining spatially positioned relative to the discretedosages of active agent.

It is also an object of the invention to provide methods for the facile,in vivo, analysis of the sensitivity of a disease or disorder in apatient to one or more active agents.

SUMMARY OF THE INVENTION

Devices for the local delivery of microdose amounts of one or moreactive agents, alone or in combination, in one or more dosages, toselected tissue of a patient are described. The devices generallyinclude multiple microwells arranged on or within a support structure.The microwells contain one or more active agents, alone or incombination, in one or more dosages and/or release pharmacokinetics.Preferably, the devices are configured to deliver the microdose amountsso as to virtually eliminate overlap in the tissue of active agentsreleased from different microwells. In certain embodiments, the devicesare configured to facilitate implantation and retrieval in a targettissue. In an exemplary embodiment, the device has a cylindrical shape,having symmetrical wells on the outside of the device, each wellcontaining one or more drugs, at one or more concentrations. The deviceis sized to permit placement using a catheter, cannula, or stylet. In apreferred embodiment, the device has a guidewire to assist in placementand retrieval. The device may also include features that assist inmaintaining spatial stability of tissue excised with the device, such asfins or stabilizers that can be expanded from the device prior to or atthe time of removal. Optionally, the device has fiber optics, sensorsand/or interactive features such as remote accessibility (such as WiFi)to provide for in situ retrieval of information and modification ofdevice release properties. In the most preferred embodiment, the fiberoptics and/or sensors are individually accessible to discrete wells.

The devices are formed of biocompatible silicon, metal, ceramic orpolymers. They may include materials such as radioopaque materials ormaterials that can be imaged using ultrasound or MRI. They can bemanufactured using techniques such as deep ion etching, nano imprintlithography, micromachining, laser etching, three dimensional printingor stereolithography. Drug can be loaded by injection of a solution orsuspension into the wells followed by solvent removal by drying,evaporation, or lyophilization, or by placement of drug in tablet orparticulate form into the wells. In a preferred embodiment, drugs areloaded on top of hydrogel pads within the microwells. The hydrogel padsexpand during implantation to deliver the drugs to the surroundingtissue. Drug release pharmacokinetics are a function of drug solubility,excipients, dimensions of the wells, and tissue into which the device isimplanted (with greater rate of release into more highly vascularizedtissue, than into less vascular tissue).

In certain embodiments, the devices are implanted directly into a solidtumor or tissue to be biopsied. Upon implantation, the devices locallyrelease an array of active agents in microdoses. Subsequent analysis oftumor response to the array of active agents can be used to identifyparticular drugs, combinations of drugs, and/or dosages that areeffective for treating a solid tumor in a patient. By locally deliveringmicrodoses of an array of drugs, the microassay device can be used totest patients for response to large range of regimens, without inducingsystemic toxicities, quickly and under actual physiological conditions.These data are used, optionally in combination with genomic data, toaccurately predict systemic drug response.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a cylindrical device containing aguidewire attached to the proximal end of the cylindrical device.

FIG. 2 is a cutaway diagram of a cylindrical device containing a fiberoptic bundle extending from the proximal end of the cylindrical device.Fiber optic elements are internally connected to each of the microwellsin the device.

FIGS. 3A-3G are schematics of an in vivo method for analyzing thesensitivity of solid tumor a patient to one or more active agents.

FIGS. 4A-D are schematics showing the arrangement of drugs in wells inthe device (FIG. 4A), implantation (FIG. 4B), dosing where drug isreleased from the wells (FIG. 4C), and the different results obtained(FIG. 4D).

FIG. 5A-5E are schematics showing testing of the device in mice.

FIG. 6 is a graph demonstrating the local concentration (mg/kg) of DrugA as a function of distance from the microwell, at three time points: 4,14 and 44 hr following in vivo implantation.

FIG. 7 shows diffusion (intensity, a.u.) of lapatinib, doxorubicin, andpaclitaxel into the tumor tissue (distance, microns) surrounding theimplanted device at 20 hours.

FIG. 8 is a cross-sectional view of the device in tissue, depictingconcentration gradient regions within a tissue for analysis of drugefficacy.

FIG. 9 depicts the concentration (mg/kg) gradient through three regions,each 100 microns from the previous region, of doxorubicin within atissue.

FIG. 10 shows the number of cleaved caspase 3 positive cells as percentarea of DAB staining as a function of distance (microns) from amicrowell in an implantable device.

FIG. 11 shows doxorubicin concentration (mg/kg) as function of distance(microns) from the microwell for 3 doxorubicin formulations (purepowder, 5% in PEG, 1% in PEG) in an A375 tumor.

FIG. 12 shows percentage of cleaved caspase 3 positive cells as afunction of distance (microns) from the microwell of an implantabledevice for 3 doxorubicin formulations, pure powder, 5% by weight in PEG1000, and 1% by weight in PEG 1000, in an A375 tumor.

FIGS. 13A and 13B show a cylindrical implantable device in whichhydrogel pads under drug to be released are used to expel compounds intosurrounding tissue as the hydrogel is hydrated following implantation,FIG. 13A at time of implantation; FIG. 13B at 4-24 hours afterimplantation.

FIG. 14 shows doxorubicin concentration (mg/kg) as a function ofdistance (microns) from the microwell of an implantable device at 4 h,14 h, and 44 h post implantation.

FIGS. 15A-15E show a minimally invasive method for analyzing thesensitivity of a solid tumor to one or more active agents. FIG. 15A isthe cylindrical device having wells for release of doxorubicin,gemcitabine, lapatinib, doxorubinc, gemcitabine, and lapatinib. FIG. 15Bis a schematic of the device implanted into a tumor, having emptied thedrugs into discrete regions of the surrounding tissue, and a coringneedle to surround and remove the device and adjacent tissue. FIG. 15Cis the device in the coring needle. FIG. 15D shows the areas of tissueadjacent to the device wells being transferred for analysis. FIG. 15Eshows the treated tissue samples to be analysed.

FIG. 16 shows a comparison of intratumor concentration (mg/kg) ofdoxorubicin following release over distance (microns) from an implanteddevice, with a polynomial curve fit, with systemic administration ofdoxorubicin.

FIG. 17 shows a comparison of intratumor concentration (mg/kg) ofdoxorubicin following release over distance (microns) from an implanteddevice as pure doxorubicin, with a polynomial curve fit, 5% doxorubicinin PEG 1450, with systemic dosing.

FIG. 18 shows differential apoptotic response (apoptotic index, percent)after device-delivery of doxorubicin in A375, BT474, and PC3 tumors.

FIG. 19 shows local tumor apoptosis (apoptotic index, percent) followingsystemic dosing in A375 and PC3 tumors.

FIG. 20 shows a differential response (apoptotic index, percent) todevice-delivery of vemurafenib in A375 and PC3 tumors.

FIG. 21 shows a differential response (apoptotic index, percent) todevice-delivery of gemcitabine in MDA-MB231 and BT474 tumors.

FIG. 22 shows a differential response (apoptotic index, percent) todevice-delivery of topotecan in PC3 and BT474 tumors.

FIG. 23 shows apoptotic response (apoptotic index, percent) to deliveryof snitinib or lapatinib from doxorubicin pre-loaded microwells in BT474tumors.

FIG. 24 shows the apoptotic response (apoptotic index, percent) todelivery of lapatinib from doxorubicin pre-loaded microwells inMDA-MB231 tumors.

DETAILED DESCRIPTION OF THE INVENTION

Devices including microwells which contain one or more active agents, inone or more different dosages, or combinations with other drugs, locallydeliver microdose amounts to discrete regions adjacent to the devicewhich can be correlated with the microwell releasing the drug or drugcombination. Loading of the microwells can be used to vary the selectionof the agent, formulation, time of release, concentration, orcombination with other actives, to discrete regions within a targettissue located proximally to the microwell. The device is removed afterdelivery, typically about 24-48 hours after implantation, along with theassociated tissue. The spatial relationship of the tissue to themicrowells is maintained during removal. Analysis of the associatedtissue allows determination of the optimal therapy for the tissue to betreated.

I. DEFINITIONS

“Microwell,” as used herein, refers to a chamber, void, or depressionformed within or on the support structure. In a preferred embodiment, itis a discrete chamber not commonly accessible via other microwells or achannel, port, or reservoir accessing more than one microwell.

“Support Structure,” as used herein, refers to the body of the device towhich one or more microwells are attached or within which one or moremicrowells are formed.

“Guidewire,” as used herein, refers to a wire-like structure attached tothe device which is intended to assist in the implantation of the deviceat a site of medical interest and/or its subsequent removal from thesite of implantation.

“Active Agent,” as used herein, refers to a physiologically orpharmacologically active agent that can act locally and/or systemicallyin the body. The term “active agent” includes agents that can beadministered to a subject for the treatment (e.g., therapeutic agent),prevention (e.g., prophylactic agent), or diagnosis (e.g., diagnosticagent) of a disease or disorder.

“Anti-neoplastic agent”, as used herein, refers to an active agent thateither inhibits the growth and multiplication of neoplastic cells, suchas by interfering with the cell's ability to replicate DNA, and/or iscytotoxic to neoplastic cells.

“Effective amount” or “therapeutically effective amount”, as usedherein, refers to an amount of one or more therapeutic agents which iseffective to decrease the size of a solid tumor or to inhibit the growthof a solid tumor.

“Biocompatible” and “biologically compatible”, as used herein, generallyrefer to materials that are, along with any metabolites or degradationproducts thereof, generally non-toxic to the recipient, and do not causeany significant adverse effects to the recipient. Generally speaking,biocompatible materials are materials which do not elicit a significantinflammatory or immune response when administered to a patient.

“Biodegradable Polymer” and “Bioerodible Polymer” are used hereininterchangeably, and generally refers to a polymer that will degrade orerode by enzymatic action or hydrolysis under physiologic conditions tosmaller units or chemical species that are capable of being metabolized,eliminated, or excreted by the subject. The degradation time is afunction of polymer composition, morphology, such as porosity, particledimensions, and environment. Suitable degradation times are from hoursto weeks, more preferable from days to weeks.

“Tumor,” as used herein, refers to an abnormal mass of tissue thatresults from the proliferation of cells. Typically, solid tumors do notcontain cysts or liquid areas within the tissue mass. Solid tumors canarise in any part of the body, and may be benign (not cancerous) ormalignant (cancerous). Most types of cancer other than leukemias canform solid tumors. Solid tumors include, for example, adenocarcinomas,carcinomas, hemangiomas, liposarcomas, lymphomas, melanomas andsarcomas.

“Tissue,” as used herein, refers to groups of cells that perform aparticular function, as well as organs, which are aggregates of tissues.

“Local Delivery” and “Local Administration,” as generally used herein,refer to the administration of an active agent to a target tissuelocation from a source that is at the target tissue location, oradjacent to or in close proximity to the target tissue location.

“Microdose,” as used herein, refers to an amount of an active agent thatis locally administered to a tissue to determine one or more clinicalparameters, such as efficacy of active agent, the metabolism of theactive agent, or a combination thereof.

“Hydrogel,” as used herein, refers to materials which swell extensivelyin water and dissolve or erode with time depending on the viscosity andthe molecular weight of the material.

“Apoptotic Index,” as used herein, refers to the percentage of apoptoticcells displaying a specific lineage antigen within a population of cellsthat remain unfragmented and retain the expression of the specificlineage antigen.

II. IMPLANTABLE DEVICES

A. Support Structure

Devices generally include one or more microwells formed on or within asupport structure. The support structure forms the body of the device.The support structure can be fabricated to form devices having a varietyof shapes. For example, the device can be cuboid, cubic, or cylindricalin shape. In the preferred embodiment, the device is cylindrical. Thesupport structure may also be configured to have one or more areas ofseparation. For example, depending on such factors as the material usedand number of microwells, the areas of separation may includeperforations, a material of enhanced flexibility or lower durometer,hinges, joints, etc., which allow portions of the support structure tobe separated or flex.

The device is preferably sized to be implanted using a needle, catheter,or surgical incision. Most preferably, the dimensions of the device aresuitable for implantation using an 18 gauge biopsy needle, stylet,cannula or catheter. In certain embodiments, the cylindrical device hasa diameter of between about 0.5 mm and about 2 mm, more preferablybetween about 0.5 mm and about 1.5 mm, most preferably between about 0.5mm and about 1.0 mm. In a particular embodiment, the cylindrical devicehas a diameter of approximately 0.9 mm. In certain embodiments, thecylindrical device has a length of less than about 5 mm, more preferablyless than about 4 mm, most preferably less than about 3 mm. In aparticular embodiment, the cylindrical device has a length ofapproximately 2.5 mm.

B. Microwells

The surface of the device includes a plurality of microwells, each ofwhich typically includes a solid bottom proximal to the supportstructure, one or more solid side walls, and an opening located on thesurface of the device distal to the support structure. Alternatively,the microwells can be in the form of a hemispherical bowl. Themicrowells must be discrete and fillable so that agent to be deliveredcan be loaded prior to implantation, but be releasable afterimplantation. The microwells may be fillable from a central lumen, orcommon delivery channel within the device, which can be directed intoone or more microwells, or filled from the outside of the device andthen sealed. In the most preferred embodiment, the microwells areisolated from other microwells to prevent any contamination of agent inone microwell with another. Microwells must be separated by sufficientsupport structure or microwell wall thickness that released agent doesnot overlap with released agent from adjacent microwells. It ispreferred not to have common connections with other microwells, butthese may be included in the event that the common connection (such as asupply channel), can be sealed at the point of entry into the microwellto prevent any cross-contamination with material from any othermicrowell.

Devices can contain any number of microwells. In the device shown in theattached figures, wells are provided in five rows of eight wells.Representative numbers of microwells range from four to about 100. Themicrowells may have any shape (e.g., circular or rectangular) anddimensions (e.g., length/width, diameter, and/or depth) suitable for aparticular application. In some embodiments, all of the microwells in adevice have the same shape and dimensions. In these cases, all of themicrowells in the device have substantially the same volume. In otherembodiments, the array contains microwells with multiple shapes,dimensions, or combinations thereof. In these cases, microwells with oneor more different volumes may be incorporated into a single device.

The microwells can have any suitable shape. For example, the microwellscan be circular, ovoid, quadrilateral, rectangular, square, triangular,pentagonal, hexagonal, heptagonal, or octagonal. In some embodiments,the microwells are rectangular in shape. In these instances, the shapeof the microwells can be defined in terms of the length of the four sidewalls forming the perimeter of the rectangular microwell.

In certain instances, the rectangular microwells have side walls rangingfrom about 50 microns to about 500 microns in length, more preferablyfrom about 100 microns to about 400 microns in length. In particularembodiments, the four side walls forming the perimeter of therectangular microwell are of substantially equivalent length (i.e., themicrowell has a square shape). Preferred sizes are 100×100, 200×200 and400×400 microns, with depths of 100 to 300 microns.

In some embodiments, the microwells are spherical in shape. In certaininstances, the spherical microwells have diameters ranging from about 50microns to about 500 microns, more preferably from about 100 microns toabout 400 microns.

The depth of the microwells, governed by the height of the solid sidewalls forming the microwells, can vary to provide microwells having thedesired volume and/or volume-to-surface-area ratio for particularapplications. In certain instances, the depth of the microwells rangesfrom about 50 microns to about 500 microns, more preferably from about75 microns to about 400 microns, most preferably from about 100 to about300 microns.

The microwells may have any volume suitable for a particularapplication. In certain instances, the volume of the microwells rangesfrom about 1.25×10⁵ cubic microns to about 1.25×10⁸ cubic microns, morepreferably from about 1.00×10⁵ cubic microns to about 6.40×10⁷ cubicmicrons, most preferably from about 1.00×10⁵ cubic microns to about4.80×10⁷ cubic microns.

The microwells may be arranged on or within the support structure in avariety of geometries depending upon the overall device shape.Preferably, the microwells are arranged so as to virtually eliminateoverlap in the tissue of active agents released from differentmicrowells. For example, in some embodiments, the microwells arearranged on or within the support structure with the axes of themicrowells relatively parallel and the distal openings in a relativelysingle plane. In this configuration the microwells can be arranged inrectangular or circular arrays. Alternatively, the microwells may bearranged in a three-dimensional pattern where the distal ends of themicrowells lie in multiple planes. In this three-dimensional pattern theaxes of the microwells may be relatively parallel or be skewed relativeto one another, depending on the overall shape of the device.

The microwells may be equally spaced from one another or irregularlyspaced. In preferred embodiments, the edges of neighboring microwellsare separated by at least about 50 microns, more preferably at leastabout 75 microns, most preferably at least about 100 microns. In certainembodiments, the edges of neighboring microwells are separated by atleast about 100 microns, about 200 microns, about 300 microns, or about400 microns.

Cylindrical devices have been manufactured with diameters ranging from500-1100 microns, with a height of 2-4 mm. Microwells have been added bymicromachining Microwell diameters ranged from 130-600 microns andmicrowell depth ranged from 50-600 microns.

Microwells may also have edges, walls, or be recessed within the deviceto help prevent overlap between agent released into the tissues from themicrowells. Means for sealing the microwells may also be designed sothat release only occurs through one area, such as the center, of themicrowell, to further limit overlap with agent released from adjacentmicrowells into the tissue.

C. Materials Used to Form Devices

Devices may be fabricated from any biocompatible material or combinationof materials that do not interfere with delivery of one or more activeagents, assays performed, or data collection, if employed.

In certain embodiments, the device is radiopaque to facilitate imagingduring implantation, residence, and/or removal. In some cases, one ormore portions of the device are fabricated from a material, such asstainless steel, which is radiopaque. In some cases, one or morecontrast agents are incorporated into the device to improve radiopacityor imaging of the device in vivo. Preferred materials includebiocompatible polymers, most preferably non-biodegradable, since thedevice is intended for removal with the adjacent treated tissue, butdegradable polymeric materials may be used to fabricate the device,allowing the device to replain in situ and efficacy of the differentagents assessed using other methodology, such as ultrasound, biopsy, orother imaging techniques.

The microwells and support structure are generally fabricated frombiocompatible materials that provide the device with suitable integrityto permit device implantation and removal, and to provide the desiredresidence time within the target tissue. In instances where themicrowells, support structure, or both are fabricated from anon-biocompatible material, the non-biocompatible material is generallycoated with another material to render the microwells and supportstructure biocompatible.

In some embodiments, the microwells and support structure are formedfrom a single material. In other embodiments, the microwells and supportstructure are formed from multiple materials that are combined so as toform an integral structure. Examples of materials that can be used toform the microwells and/or support structure include polymers,silicones, glasses, metals, ceramics, inorganic materials, andcombinations thereof. In certain embodiments, the microwells and supportstructure are formed from composite materials, such as, for example, acomposite of a polymer and a semiconductor material, such as silicon.Devices have been manufactured out of the following materials, Acrylicresin, polycarbonate, Acetal resin (DELRIN®), polytetrafluoroethylene(TEFLON®, polyether-ether-ketone (PEEK), polysuflone and polyphenolsulfone (RADEL®).

In some embodiments, the microwells, support structure, or combinationthereof, are formed from or include a polymer. Examples of suitablepolymers include polyacrylates, polymethacrylates, polycarbonates,polystyrenes, polyethylenes, polypropylenes, polyvinylchlorides,polytetrafluoroethylenes, fluorinated polymers, silicones such aspolydimethylsiloxane (PDMS), polyvinylidene chloride,bis-benzocyclobutene (BCB), polyimides, fluorinated derivatives ofpolyimides, polyurethanes, poly(ethylene vinyl acetate), poly(alkyleneoxides) such as poly(ethylene glycol) (PEG), or copolymers or blendthereof.

In certain embodiments, microwells, support structure, or combinationthereof, are fabricated from or include one or more biodegradablepolymers. Examples of suitable biodegradable polymers includepolyhydroxyacids, such as poly(lactic acid), poly(glycolic acid), andpoly(lactic acid-co-glycolic acids); polyhydroxyalkanoates such aspoly3-hydroxybutyrate or poly4-hydroxybutyrate; poly(caprolactones);poly(orthoesters); poly(phosphazenes); polyesteramides; polyanhydrides;poly(dioxanones); poly(alkylene alkylates);poly(hydroxyacid)/poly(alkylene oxide) copolymers;poly(caprolactone)/poly(alkylene oxide) copolymers; biodegradablepolyurethanes; poly(amino acids); polyetheresters; polyacetals;polycyanoacrylates; poly(oxyethylene)/poly(oxypropylene) copolymers, ora blend or copolymer thereof, may be used. Biodegradable shape memorypolymers, such as those described in U.S. Pat. No. 5,189,110 or U.S.Pat. No. 5,139,832, may also be employed.

In some embodiments, the microwells, support structure, or combinationthereof, formed from or include a metal. Examples of suitable metalsinclude, but are not limited to, cobalt, chromium, nickel, platinum,gold, silver, silicon, stainless steel, titanium, tantalum, and any oftheir alloys (e.g., nickel-titanium alloys), and combinations thereof.Biodegradable metals such as magnesium-based metals may also be used.

In particular embodiments, the microwells, support structure, orcombination thereof are fabricated from or include silicon or a ceramicsuch as hydroxyapatite. In particular embodiments, the microwells,support structure, or combination thereof are fabricated from or includea polymer formed from SU-8, the structure of which is shown below.

The device may include an agent that prevents or reduces biofilmformation or inflammation or other foreign body reaction to the deviceonce implanted. Such an agent may be incorporated within one or more ofthe component materials of the device, or coated on a surface thedevice, or portions thereof. In certain embodiments, one or moreportions of the device is coated with a polymer coating to prevent orreduce biofilm formation or inflammation or other foreign body reactionto the device.

Preferably, the device is cylindrical in shape to facilitateimplantation and minimize tissue damage. A representative example of acylindrical device is illustrated in FIG. 1. The device 10 contains asupport structure 16, forming the body of the device. The device has aproximal end 14 and a proximal end 12, from which a guidewire 20extends, and a plurality of microwells 18 formed within the supportstructure. One or more of the microwells contain an active agent oragents 22, which can be released independently or in combination.

Preferably, the device is formed of Acetal resin (DELRIN®,polyether-ether-ketone (PEEK), polysuflone or polyphenol sulfone(RADEL®)s which has the advantages of being biocompatible, resistant tofracturing, easily manufactured with high resolution) or SU8polyethylene, which has the advantage of being very biocompatible, andsofter, thereby allowing microtome sectioning.

D. Guidewires

In some embodiments, the device also includes a guidewire designed toassist in the implantation of the device at a site of medical interestand/or its subsequent removal from the site of implantation. Theguidewire may be attached to or extend from any portion of the device.In certain embodiments, the guidewire extends from the proximal end ofthe device.

The guidewire can be any wire-like structure dimension and length whichis suitable to assist in the implantation of the device at a site ofmedical interest and/or its subsequent removal from the site ofimplantation. In certain embodiments, the guidewire has a diameter ofbetween about 0.010 inches and about 0.065 inches. The length of theguidewire typically ranges from about 30 cm to about 300 cm (or more) inlength; however, the guidewire is typically long enough to extend fromthe site of device implantation to a point outside of the patient'sbody, such that the guidewire remains externally accessible afterimplantation of the device.

Guidewires can be fabricated from any material or combination ofmaterials, such as polymers, metals, and polymer-metal composites.Examples of suitable materials include metals, such stainless steel(e.g., 304 stainless steel), nickel and nickel alloys (e.g., NITINOL® orMP-35N), and cobalt alloys, polymers, such as polyurethanes, elastomericpolyamides, block polyamide-ethers, and silicones. Radiopaque alloys,such as platinum and titanium alloys, may also be used to fabricate, inwhole or in part, the guidewire.

In certain embodiments, the guidewire is coated or treated with variouspolymers or other compounds in order to reduce foreign body reactionprovide or to provide desired handling or performance characteristicssuch as to increase lubricity. In certain embodiments, the guidewire iscoated with polytetrafluoroethylene (PTFE) or a hydrophilic polymercoating, such as poly(caprolactone), to enhance lubricity and impartdesirable handling characteristics to the guidewire.

E. Sensors and Fiber Optics

In some embodiments, the device also includes a fiber optic bundle, orother interrogatable or addressible means extending from a portion ofthe microassay device, and/or sensors which are attached to or insertedwithin the microwells, to provide feedback while implanted or afterretrieval of the device. These may also be used to trigger release ofthe active agent.

The length of the fiber optic bundle typically ranges from about 30 cmto about 300 cm (or more) in length; however, the fiber optic bundle istypically long enough to extend from the site of device implantation toa point outside of the patient's body, such that the fiber optic bundleremains externally accessible after implantation of the device.

In these embodiments, individual fiber optic elements within the fiberoptic bundle may be internally wired to one or more of the microwells inthe miroassay device. The fiber optic elements can be interfaced withexternal signal processing means to analyze the contents of themicrowells, the nature of tissue proximal to the microwells, andcombinations thereof. The fiber optic elements can also be interfaceswith an external energy source to trigger the release of a drug or toprovide photodynamic therapy.

The interrogatable means may be connected to sensors adjacent to orwithin the microwells. These may also have means for remote accessing,such as a WiFi connection.

Integrated optical fibers can provide real-time sensing of drug effect.In a preferred embodiment, optical fibers 12-250 micron in diameter areintegrated into a cylindrical device. These fibers enable local sensingof the effect of released compound on the tissue adjacent to themicrowell. They can be used to measure specific changes in tissuecharacteristics that represent biological alterations in tissue state,e.g. apoptosis.

FIG. 2 illustrates a cylindrical device containing integrated fiberoptic components. In this embodiment, the device 30 contains a supportstructure 36, forming the body of the device. The device has a distalend 34 and a proximal end 32, from which a fiber optic bundle 40extends, and a plurality of microwells 38 formed within the supportstructure. Individual fiber optic elements within the fiber optic bundleare internally wired to the microwells in the miroassay device.

F. Tissue Retainers

In some embodiments, the device also contains a feature, such as anoverhang or lip, to facilitate the removal of a tissue sampleimmediately surrounding the device upon device removal. The device mayalso include retainers that are recessed into the device untilimplantation or removal. These are then expanded outwardly into thetissue where they can serve to stabilize or maintain the spatialarrangement of the tissue relative to the device and/or decrease anyoverlap in drug diffusion between wells.

The device can also contain a fastening means, such as a snap-lockfastener, or a magnet at the proximal end of the device to facilitatedevice removal.

G. Active Agent Release Mechanisms

Drug compounds have inherently different transport rates, which dependon their chemical properties. To obtain optimal diffusion of the activeagent into the surrounding tissue, one option is to control the releaseof the active agent from the microwells. Preferably, release of theactive agent is controlled so as to virtually eliminate overlap in thetissue of active agents released from different microwells. The releasesystems may be natural or synthetic. In some variations, the releasesystem may be selected based on the period over which release isdesired, the rate of diffusion desired, or the amount of diffusiondesired. Active agents from microwells can be released not only withdistinct active agents and concentrations, but also at differentkinetics, depending on (potentially) a different material coating ineach well (such as platinum or gold or polymer).

i. Microwell Opening

Altering the size of the microwell opening can control the rate of drugrelease. A large opening results in a faster release of the active agentinto the surrounding tissue than a small opening. This may beadvantageous for drugs that diffuse slowly through the tissue. A smalleropening may be advantageous for drugs that diffuse rapidly through thetissue.

ii. Membranes and Films

A membrane or film may be applied to the well after the active agent isincorporated to isolate the active agent until the time of use. The filmmay be manually removed immediately prior to use or may be degraded uponimplantation to allow release of the active agent into the surroundingtissue. Alternatively, a porous membrane may be used to cover themicrowells to control rate of release after implantation.

iii. Matrices

The active agent may be contained within a matrix formed of abiodegradable material or a material which releases the incorporatedsubstance by diffusion out of or degradation of the matrix, or bydissolution of the substance into surrounding interstitial fluid.Preferably, the matrix includes poly (ethylene glycol) (PEG). Whenprovided in a matrix, the substance may be homogeneously orheterogeneously distributed within the matrix. Selection of the matrixmay be dependent on the desired rate of release of the substance. Bothbiodegradable and nonbiodegradable matrices can be used for delivery ofthe substances. Suitable release matrices include, without limitation,polymers and polymeric matrices, non-polymeric matrices, or inorganicand organic excipients and diluents such as, but not limited to, calciumcarbonate and sugar.

iv. Hydrogels

As shown in FIG. 13A, a hydrogel pad can be placed within each microwell72. Compounds 70 may then be placed on top of hydrogel pads 71 locatedwithin the microwells 72. As shown in FIG. 13B, when the device 73 isimplanted, small amounts of fluid from the surrounding tissue diffuseinto the microwells 72 and cause the hydrogel pads 71 to expand. Duringexpansion, the compounds 70 are forced into the surrounding tissue.Hydrogel release mechanisms can achieve significantly larger intratumoractive agent concentrations in short time frames and therefore allowsfor more a rapid active agent efficacy analysis.

Active agent delivery can be fine-tuned by using hydrogels withdifferent hydrophilic expansive properties. Preferably the hydrogel ispoly-acrylamide based. Other exemplary hydrogel-forming polymermaterials include, but are not limited to, cellulose ethers, preferablydifferent viscosity/molecular weight grades of hypromelloses such ashydroxypropyl methyl cellulose (HPMC K4M to K100M available from DowChemical); cross-linked acrylates such as CARBOPOL®; alginates; guar orxanthan gum; carrageenan; carboxymethylcellulose; and mixtures thereof.The hydrogel-forming polymeric material is present in an amount fromabout 2% to about 80% by weight, preferably 3% to 50% by weight of thematrix.

III. ACTIVE AGENTS

One or more active agents are incorporated in one or more of themicrowells in the devices. In some devices, the microwells contain oneor more active agents, in one or more dosages, alone or in one or morecombinations. In other devices, not all of the microwells contain anactive agent. In these embodiments, empty microwells may serve as acontrol, or increase distance between released active agents to decreaseor eliminate overlap of diffused drug.

In some embodiments, each microwell which contains an active agentcontains a different active agent or different combination of activeagents. In some embodiments, the microwells each contains an activeagent or combination of active agents in differing amounts of activeagents, differing ratios of active agents, or differentexcipients/formulations of active agents. This allows variation not onlyof the drug, but also the dosage, release pharmacokinetics, and testingof various combinations at the same.

A. Compounds

In preferred embodiments, the active agent is an anti-neoplastic agent.Representative anti-neoplastic agents include, but are not limited to,alkylating agents (such as cisplatin, carboplatin, oxaliplatin,mechlorethamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine,carmustine, procarbazine, chlorambucil and ifosfamide), antimetabolites(such as fluorouracil (5-FU), gemcitabine, methotrexate, cytosinearabinoside, fludarabine, and floxuridine), antimitotics (includingtaxanes such as paclitaxel and decetaxel and vinca alkaloids such asvincristine, vinblastine, vinorelbine, and vindesine), anthracyclines(including doxorubicin, daunorubicin, valrubicin, idarubicin, andepirubicin, as well as actinomycins such as actinomycin D), cytotoxicantibiotics (including mitomycin, plicamycin, and bleomycin), andtopoisomerase inhibitors (including camptothecins such as camptothecin,irinotecan, and topotecan as well as derivatives of epipodophyllotoxinssuch as amsacrine, etoposide, etoposide phosphate, and teniposide).Anti-angiogenic compounds may also be tested, such as thalidomide.

Other active agents may be anti-infectives such as antivirals,antibiotics, or antifungals; or immunomodulators, such asimmunoenhancers, vaccines, or immunosuppressants (includinganti-inflammatories); or hormones or their analogues, or hormoneagonists or antagonists.

Active agents may be small molecule active agents or larger molecules(e.g., macromolecules) such as proteins, peptides, carbohydrates andnucleic acids. “Small Molecule”, as used herein, refers to a molecule,such as an organic or organometallic compound, with a molecular weightof less than 2,000 Daltons, more preferably less than 1,500 Daltons,most preferably less than 1,000 Daltons. The small molecule can be ahydrophilic, hydrophobic, or amphiphilic compound.

B. Microdose

The devices deliver a microdose amount of a substance to a targettissue. A microdose amount may be from about 0.001 μg (or less) to about1,000 μg, or about 10,000 μg (or more) of the substance. Preferably, theamount of the microdoes is optimized so as to virtually eliminateoverlap in the tissue of active agents released from differentmicrowells. Those of skill will readily appreciate that microdose levelsmay vary as a function of the specific substance employed, the targettissue, and/or the medical condition being treated. Appropriate dosesmay be determined as described in example 1.

The compound may be delivered in a controlled release, sustainedrelease, delayed release, bolus followed by sustained release, and/orpulsatile release. Delivery may also occur over any time period. Forexample, it may occur over a period of minutes to hours, or days toweeks. In the preferred embodiment, release is complete within 48 hours,with substantially all drug being released within 12, 24, 36, or 48hours. Preferably, the release profile and delivery time is optimized soas to virtually eliminate overlap in the tissue of active agentsreleased from different microwells.

The drug may be applied as a powder, particulate, or in a solution orsuspension, with the solvent removed by drying, evaporation,lyophilization or suction.

IV. METHODS OF MANUFACTURE

Devices can be fabricated using methods known in the art, such aspatterning, photolithography, etching and CNC micromachining. Suitablemethods for the manufacture of devices can be selected in view of avariety of factors, including the design of the device (e.g., the sizeof the device, the relative arrangement of device features, etc.) andthe component materials used to form the device.

Examples of suitable techniques that can be used, alone or incombination, for the fabrication of devices include LIGA (LithographicGalvanoforming Abforming) techniques using X-ray lithography,high-aspect-ratio photolithography using a photoresist, such as anepoxy-based negative photoresist such as EPON™ SU-8 (also referred to asEPIKOTE™ 157), microelectro-discharge machining (μEDM),high-aspect-ratio machining by deep reactive ion etching (DRIE), hotembossing, 3-dimensional printing, stereolithography, laser machining,ion beam machining, and mechanical micro-cutting using micro-tools madeof hard materials such as diamond.

Detailed methods for microfabrication are described in, for example,“Microreactors, Epoch-making Technology for Synthesis” (edited byJun-ichi Yoshida and published by CMC Publishing Co., Ltd., 2003) and“Fine Processing Technology, Application Volume—Application toPhotonics, Electronics and Mechatronics—” (edited by the MeetingCommittee of the Society of Polymer Science, Japan, and published by NTSInc., 2003.

Devices have been loaded with distinct compounds in up to 30 microwells.The compounds have been loaded as crystalline powder, lyophilizedpowder, compressed microtablets, as liquids dissolved in water or buffersolution, as solid dissolved in poly(ethylene-glycol) of molecularweight 200, 400, 600, 800, 1000, 1450, 3400 and 7500.

V. METHODS OF USE

The device and corresponding assays deliver confined precise quantitiesof drugs into solid tissue within a living organism and allow rapid andminimally invasive diagnostic assessment of in vivo interactions betweendrugs and tissues. Instead of removing cells or tissue out of theirnative environment for ex vivo analyses, the device and correspondingassays allow for in vivo assessment of local drug efficacies in thenative microenvironement. Further, the device can locally delivercompounds to adjacent tissue and achieve tissue concentrations thatcorrespond or are equivalent to tissue concentrations achieved bysystemic dosing. Therefore, the device and corresponding assays providephenotypic information on drug-tissue interaction in a rapid,high-throughput, and minimally invasive way with no systemic effects.

The device is implanted directly into a tumor or other tissue to betreated. The tissue will typically be transformed, i.e. canceroustissue, but may also be infected with bacteria, fungus or virus, in needof immunomodulation (i.e., immunosuppression or immunoenhancement), orin need of hormonal adjustment. In some cases the hormone may be usefulfor treating a cancer. The device is particularly useful in treatingrefractory disorders and in testing combination of drugs that may bemore effective in combination.

The device releases an array of drug microdoses locally, and uses stateof the art detection methods to identify the drugs or combinationsinducing a response. By using microdoses of drugs, the device is capableof testing each patient for response to large range of regimens, withoutinducing systemic toxicities. These data can be used along with genomicdata to accurately predict systemic drug response.

In some variations, a microdose amount is used in early human studies,e.g., before a phase I clinical trial, to evaluate the effect of thesubstance on a target tissue, or to obtain pharmacokinetic or metabolicdata. In other variations, a microdose amount is used to locally treat amedical condition, e.g., a cancer or tumor. In yet other variations, amicrodose amount is used to locally deliver a contrast agent for astructural or functional imaging procedure. In view of this, a microdoseamount can be tailored to the specific indication of the substancedelivery.

The assay may be used to detect one or more of: a degree of agentpermeation through the target tissue; detect a physiochemical effect ofthe agent on the target tissue; and detect a pharmacological effect ofthe agent on the tissue. In further variations, the devices may includea sensor for sensing one or more parameters of the target tissue afterdelivery of the substance. An agent may be delivered as a result of theresponse parameter or in response to the data obtained by the assayand/or sensor. The assay may be configured to provide various data suchas data related to efficacy such as chemotherapeutic efficacy; activitysuch as tumor cell invasiveness; toxicity such as toxicity due to one ormore agents being delivered or toxicity due to cell death; andcombinations of these.

Methods have been developed for integrating antibody coatings into thedevice with the goal of capturing the presence of biomarker proteins inthe local tissue near a microwell. Biomarkers can then bind to thespecific antibody coating and remain tethered to the device. In such ascenario, the device is pulled out from the tissue following the desiredincubation time, and biomarker concentrations are determined ex-vivodirectly by examination of the device.

A. Target Tissues

The target tissue may be located anywhere in the patient's body such aslocations including: liver, lung, kidney, prostate, ovary, spleen, lymphnode, thyroid, pancreas, heart, skeletal muscle, intestine, larynx,esophagus and stomach. In a preferred embodiment, the target tissue istumor tissue such as adenoma, adenocarcinoma, squamous cell carcinoma,basal cell carcinoma, small cell carcinoma, large cell undifferentiatedcarcinoma, chondrosarcoma, fibrosarcoma, and combinations thereof.

The target tissue may also be a tissue which is infected, for example,with a virus, bacteria, fungus or parasite, or which is characterized byinflammation or is in need of immunostimulation.

B. Delivery and Retrieval of the Device

Devices may be implanted via percutaneous, minimally invasive, or openprocedures into the tissue of a patient. For example, devices may bedelivered via an open surgical procedure, or by a minimally invasiveprocedure such as laparoscopy, endoscopy, arthroscopy, andcatheter-based procedures. The devices may also be deliveredpercutaneously, for example using a needle, such as a 19 to 24 gaugebiopsy needle. Retrieval of the devices may occur via the sameprocesses, typically also using a biopsy needle with but with a largerdiameter, such as a 13, to 18 gauge needle. The inserting needle is acutting needle that has a smaller diameter than the retrieval needle,which is a larger diameter coring needle.

An image of the target tissue, such as a tumor, may be performed priorto implantation, during implantation, during implant residence, duringimplant removal, after implant retrieval, and combinations thereof. Incertain embodiments, the microassay device is implanted in the patientwith image guidance.

In most cases, the device is implanted into a tumor using a biopsy-typeneedle, cannula, catheter or stylet. The device can also be placed in alumen, such as a bile duct, alveoli or bronchi or kidney tubule.Alternatively, the device can be placed during a procedure such as abiopsy or excision of tumor.

In the preferred embodiment, the device is placed using a cutting biopsyneedle with sharp stuffer tip. The stuffer needles are then retractedwhile keeping the needle in place. The device is delivered through theneedle, then the need is retracted. A guidewire may be attached prior toor at the time of implantation. The advantage of this method is thatthere is better tissue penetration into the wells, and less tissueinjury.

The device is retrieved in conjunction with the adjacent tissue. Thegoal is to analyze the tissue in the spatial orientation relevant to thedevice, to allow assessment of efficacy, dose dependency, and type ofresponse (i.e., apoptosis, necrosis, inflammation, subclinicalresponse). In a preferred embodiment, the device is retrieved byexcising the device and associated tissue at one time, for example, bycutting out the device with a uniform amount of tissue around thedevice. In the case of a cylindrical device, one excises the deviceusing a cutting needle, coring biopsy needle, or catheter that is of agreater diameter than the device. The guidewire may be used to insurethat the tissue remains placed in the same proximity to the device.Stabilizers or retainers may be used in either the cutting removaldevice or the implanted device to help maintain spatial relationshipwith the device and treated tissue.

C. Analysis of Tissue

Following retrieval, usually less than 7 days from implantation, morepreferably within 24 to 48 hours following implantation, the treatedtissue samples are analyzed, for example, by microscopic examination, byenzyme assays, and other histology and immunohistochemistry techniquesused to assess cancer or infected cells.

FIG. 3 illustrates an in vivo method for analyzing the sensitivity of asolid tumor of a patient to one or more active agents. An 18 g cuttingbiopsy needle 51 with stylet 52 is inserted into a solid tumor 50. Thestylet 52 is retracted, leaving the needle 51 in place. The stylet 52 isused to push the device 53 into the tumor 50. The device 53 remains inthe tumor 50 except for a retrieving device 54. A larger (14 gauge)coring needle 55 is inserted into the tumor 50 around the device 53. Theneedle 55 is retracted, taking the device 53 and surrounding tissue 56.The device 53 is then embedded in acrylic 57, sectioned and histologypreformed.

FIGS. 15A-15E depict another minimally invasive method for analyzing thesensitivity of a solid tumor to one or more active agents. Animplantable device 80 with several drug microwells 81 (FIG. 15A) isimplanted into a tissue, such as a tumor tissue 82, using a small biopsy(e.g. 18 gauge) needle. Preferably, the drug microwells 81 are locatedon opposite sides of the implantable device 80. The device is left insitu for a suitable amount of time. Typically, the device is left insitu for 12-72 hours. A larger (e.g. 12 gauge) coring needle 83 isinserted into the tumor at the time of tumor removal (FIG. 15B). Thecoring needle 83 is precisely positioned concentric with the long axisof the device by ultrasound, computed tomography, or stereotactictechniques, including use of a guide wire implanted with the device. Thecoring needle 83 carves a cylinder around the device 80, removing thedevice 80 and a cylinder of tissue 84 (FIG. 15C). The thickness of thecylinder of tissue 84 removed is dependent upon the gauge of the coringneedle 83. Typically, cylinder of tissue 84 is approximately 500 μmthick. The cylinder of tissue 84 is immediately interfaced withimplantable device 80. Ex vivo, the cylinder of tissue 84 is cut openand flattened into a slab of tissue 85 (FIG. 15D). As shown in FIG. 15E,this leaves the contents 86 from the drug microwells aligned in the sameplane. The flattened tissue slab can then be analyzed byimmunohistochemistry and other techniques. In some embodiments, the slabof tissue is embedded in paraffin, acrylamide or other fixationcompounds in preparation for preservation or analytical techniques.

FIGS. 4A-D are schematics showing the arrangement of drugs in wells inthe device (FIG. 4A), implantation (FIG. 4B), dosing where drug isreleased from the wells (FIG. 4C), and the different results obtained(FIG. 4D).

VI. KITS

Kits may contain one or more of the devices described above. Any numberand type of deployment tools, retrieval tools, and imaging devices mayalso be included. The kits may also contain additional in vitro assaysfor evaluating samples, such as a matrix for fixing tissue samples forfuture histological analysis.

The kits may also include instructions for using the devices, tools,and/or assays contained therein.

EXAMPLES Example 1 Prototype Testing in Mouse Model

Materials and Methods

As shown in FIG. 5, a mouse model for a human cancer cell line isprepared by injection of human cancer cells such as MDA MB-231 into themammary fat pad of an immunodeficient mouse. Tumors are allowed toimplant and proliferate to approximately 150-170 mm³.

Individual drugs are administered systemically by injection to the miceto establish local pharmacokinetics for the drugs. For breast cancercells, representative drugs to be tested include docetaxel, doxorubicin,irinotecan, transtuzumab, and bevacizumab.

Devices were tested in approximately 50 animals for biocompatibility andintegration with tissue. Data was obtained by computed tomography,magnetic resonance and histopathology.

A device with 14 microwells was loaded with approximately 1.5 microgramdoxorubicin (crystalline powder) per microwell. The device can be loadedwith the same drugs based on the results of the systemic testing. Eachdrug is loaded separately and in more than one concentration, as well asin combination. After 12, 24, 36 and 48 hours, devices were removed andhistology of the tissue was examined to determine the effect of thecompounds on the tumor cells adjacent to each well.

The effects of compounds eluted from microwells can be assessed bydifferent techniques. Tissue excised with the device can be assayed bystandard histopathological techniques, including immunohistochemistryand immunofluorescence. Mass spectrometry may also be used to measurelocal biomarkers indicative of an effect of a compound.

Analysis for apoptosis, necrosis, mitotic cell death, and proliferationcan also be conducted. The local microdose response was then determinedand used to define an appropriate therapeutic regime for the cancer.

Results

Computed-tomographic images of the device implanted in tumor tissueshowed microwells filled with nanoparticle compound.

Images from histopathological analysis of cross-sections of excisedtumor tissue with the implantable device show ingrowth of tissue intodevice microwell. Ingrowth of tissue, ranging from 20 to about 300microns, can be visualized by staining tissue/device section by standardimmuno-histochemistry (IHC) techniques, including Hematoxylin&eosin(H&E) staining, or any nuclear cell stain such as DAPI.

Example 2 Methods for Controlled Local Release of Drugs into Tissue

Materials and Methods

Several methods for controlling the release/diffusion of compounds intotissue, including precise spatial placement of microwells along devicemantle; geometry and size of microwells; and formulation of releasedcompounds, were developed. In this manner, the device microwells fromwhich the compounds diffuse are engineered to expose only regions oftissue that are directly adjacent to the microwell opening, to thereleased compound. This creates distinct local regions in the tissue inwhich the effect of compounds can be assessed without interference ofother compounds released from different microwells. Creation of discreteareas of drug is extremely important if one is to assess the efficacy ofthe different agents, or combinations thereof, and/or dosages and/ortimes of release (sustained, pulsed, delayed, bolus followed bysustained, etc.).

Results

Cross-sectional images of tissue surrounding the device show release oftwo compounds. Drug A was released upward and diffused into a largerregion, while Drug B was released downward into a relatively smallerregion.

The precise control over the transport time as a function of distancefrom microwells is shown in FIG. 6, demonstrating the localconcentration of Drug A as a function of distance from the microwell, atthree time points following in vivo implantation.

Example 3 Defined and Segregated Release of Multiple Compounds fromAdjacent Microwells

Materials and Methods

As in Example 2, different compounds were loaded into individualmicrowells in different formulations in order to control the rate ofrelease of compounds into the tissue. Here, doxorubicin, lapatinib, andpaclitaxel with distinct molecular weights (544 g/mol, 581 g/mol, and854 g/mol, respectively) and physical properties were loaded into thedevice. The device was implanted into tissue. The device and surroundingtissue was removed twenty hours post-in vivo implantation. Diffusion ofthe compounds from the device was evaluated twenty hours post-in vivoimplantation.

Results

Fluorescent imaging of cross-sections of the excised surrounding tissueshowed diffusion of the compounds doxorubicin, lapatinib, and paclitaxelfollowing in vivo implantation for twenty hours. Cross-sectionalfluorescent imaging showed each compound being confined to the tissue insegregated regions corresponding to where the microwell containing agiven compound was located. There was no significant overlap of drugswithin the surrounding tissue, thus demonstrating segregated diffusionof the compounds within the tissue.

FIG. 7 shows diffusion of compounds into the tumor tissue surroundingthe implanted device. Diffusion is shown by local fluorescent intensityof the drugs as a function of distance from the microwell. At twentyhours post-in vivo implantation, each drug migrated different distancesfrom their respective microwells. For at least doxorubicin andlapatinib, compound concentration decreased with increased distance fromthe microwell.

Example 4 Efficacy of Device-Delivered Compounds within Local Tissues

Materials and Methods

Doxorubicin was loaded into a microwell in the implantable device. Thedevice was implanted into tumor tissue. This was repeated for threedifferent murine human cell tumor models (BT474, PC3, A375). Afterimplantation for 20 hours, the device and surrounding tissue wasremoved, and embedded in acrylic for cross-sectioning andhistopathological analysis.

Fluorescent imaging techniques were used on sample cross-sections todetermine the doxorubicin concentration and diffusion profile within thetissue surrounding the device. Standard histopathological techniques,such as but not limited to, immunochemical techniques, can be used tomeasure a desired characteristic within the surrounding tissue todetermine effectiveness of a compound. In this instance, cleaved caspase3 antibodies were used to detect cells undergoing apoptosis.

Efficacy of doxorubicin at different concentration gradients within thetissue was quantified using a two-step analysis. First, the tumor wasdivided into regions based on distance from the microwell and drugconcentration. As shown in FIG. 8, tumor sections containing thediffused drug were then divided into regions based on concentrationgradient of the drug. The regions are aligned on a line profileextending radially out from the microwell. The first region, whichcorresponds to the greatest concentration gradient, extendsapproximately 100-150 μm from the microwell into the surrounding tissue.The next region begins approximately where the first region ends andextends another 100 to 150 μm into the surrounding tissue. The thirdregion begins approximately where the second region ends and extends100-150 μm into the tissue. Thus, each region corresponds to differentconcentration gradients of doxorubicin within the tissue.

Second, the effect of the drug on a measurable characteristic in eachconcentration region is determined. In this example, the amount of cellsundergoing apoptosis, as determined by cleaved caspase 3 antibodybinding, was evaluated in each doxorubicin concentration region.Efficacy of the doxorubicin released from the microwell is determined byantibody staining of tissue sections divided into regions correspondingto local compound exposure.

Results

FIG. 9 depicts the concentration gradient regions of doxorubicin withina tissue. Three concentration gradient regions were defined (dashedboxes). As the distance from the microwell increases, the concentrationof the compound decreases. FIG. 10 shows the number of cleaved caspase 3positive cells as percent area of 3,3′-diaminobenzidine (DAB) stainingas a function of distance from the microwell. Doxorubicin had differenteffects within the tumor types and caused the greatest number of cellsto undergo apoptosis in the A375 tumor model. This effect was seen at adistance of approximately 100-250 μm from the microwell (concentrationgradient region 2).

Example 5 Use of Multiple Biomarkers for Complete Analysis of DrugEfficacy

Anti-cancer agents inhibit tumor growth by different mechanisms.Therefore, a combination of biomarkers may be needed to fully understandthe effect of a given drug.

Materials and Methods

Doxorubicin was loaded into microwells in the device and implantedwithin a BT474 tumor. After 20 hours, the device and surrounding tissuewas removed. Protein expression of cleaved caspase 3 andPoly(ADP-ribose) polymerase (PARP) was evaluated by immunohistochemicalanalysis. These biomarkers are indicators of apoptosis. Proteinexpression of Ki67 and survivin was evaluated by immunohistochemicalanalysis. Ki67 is a biomarker for monitoring reduced cell proliferationrates. Survivin is a biomarker for monitoring reduced inhibition ofapoptosis.

Effect of 20 hour doxorubicin exposure on the expression of thebiomarkers was determined as described in Example 4. However, here fourconcentration gradient regions of approximately 100 μm were determinedand evaluated.

Results

Cleaved caspase 3 expression was greatest at a distance of approximately150-250 μm away from the microwell. Ki67 expression was greatest in theregion farthest from the microwell (approximately 350-450 μm from themicrowell). However, Ki67 expression decreased in the regionapproximately 250-350 μm away from the microwell. Ki67 expressioncontinued to decrease to undetectable levels in the region closest tothe microwell. Survivin expression was greatest in the two regionsfarthest from the microwell (approximately 250-450 μm from themicrowell). Like Ki67, survivin expression decreased in the regionspanning approximately 150-250 μm from the microwell. Survivinexpression was undetectable in the region closest to the microwell.

Example 6 Dose Ranging of Delivered Compounds and Agents

In some cases it is important to control compound concentration. Thisexample demonstrates control of drug concentration by dissolving thecompound in poly (ethylene-glycol) (PEG).

Materials and Methods

Varying percentages (1% and 5% by weight) of doxorubicin was dissolvedin PEG having a molecular weight of 1000 or 1450. Pure powderdoxorubicin and the PEG-doxorubicin formulations were loaded intomicrowells in the device. The device was implanted intotumors. After 20hours, the device and surrounding tissue was removed and analyzed fordoxorubicin concentration and cleaved caspase 3 expression, aspreviously described.

Results

FIG. 11 shows doxorubicin concentration as a function of distance fromthe microwell for 3 doxorubicin formulations in an A375 tumor. Tissueconcentration of all doxorubicin formulations was greatest in theregions closest to the microwell. Tissue concentration of alldoxorubicin formulations decreased as distance from the microwellincreased. Doxorubicin concentration was greatest in the regionsevaluated when delivered in a pure powder. Doxorubicin concentration inall regions was reduced by dissolving doxorubicin in PEG 1000 prior todelivery. Further, the distance that doxorubicin diffused into thesurrounding tissue decreased by dissolving doxorubicin in PEG 1000 priorto delivery.

FIG. 12 shows percentage of cleaved caspase 3 positive cells by distancefrom the microwell for 3 doxorubicin formulations in an A375 tumor. Thegreatest percentage of cleaved caspase 3 positive cells was observed inthe region approximately 100-250 μm from the microwell. There was aformulation dependent response. The pure powder formulation resulted inthe greatest percentage of cleaved caspase 3 positive cells. The 1% PEG1000 formulation resulted in the least percentage of cleaved caspase 3positive cells. The 5% PEG 1000 formulation produced an intermediateresponse.

FIG. 17 shows a comparison of intratumor concentration of doxorubicinfollowing release from an implanted device as pure doxorubicin, with apolynomial curve fit, 5% doxorubicin in PEG 1450, and systemic dosing(As described in Example 8). Maximal and average doses followingsystemic dosing are also shown in FIG. 17. Similar to the results afterdelivery of 5% doxorubicin in PEG 1000, 5% or 10% doxorubicin in PEG1450 lowered the local concentration of drug in the affected tumorregion.

Example 7 Effect of Implantation Time on Tissue Concentration ofDoxorubicin

The action of chemotherapeutic drugs is often concentration dependent.Exposure time of the tissue to the device can affect the concentrationof drug within the surrounding tissue. This example shows the effect ofdevice implantation time on local drug concentration within thesurrounding tissue.

Materials and Methods

Microwells were loaded with pure doxorubicin and the device wasimplanted into tumor tissue. The device and surrounding tissue wasremoved at varying times post-implantation. The concentration ofdoxorubicin in the surrounding tissue was evaluated usingimmunohistochemical techniques and fluorescent imaging.

Results

Microwells loaded with pure doxorubicin release drug into thesurrounding tumor tissue upon implantation, resulting in a steepgradient of drug concentrations. FIG. 14 shows doxorubicin concentrationas a function of distance from the microwell at 4 hours, 14 hours, and44 hours post implantation. Cross-sectional analysis of the surroundingtissue at 20 hours post implantation showed that at distances 0-130 μmfrom the microwell, concentration of doxorubicin concentration wasapproximately 15-20 mg/kg. Concentration of doxorubicin wasapproximately 8-13 mg/kg at a distance of approximately 130-200 μm fromthe microwell. Concentration of doxorubicin was approximately 3-7 mg/kgat a distance of approximately 200-300 μm from the microwell.

Example 8 Direct Comparison of Intratumor Concentration of DoxorubicinDelivered Locally by the Device or by Systemic Administration

The action of chemotherapeutic drugs is often concentration dependent.When inferring sensitivity of a tumor to a given drug, it is preferablethat the local concentration of the drug released from a microwell onthe device matches concentration levels achieved within the tumor aftersystemic dosing. This example demonstrates that local delivery by thedevices described can achieve intratumor concentrations that are theequivalent of those achieved with systemic dosing.

Materials and Methods

Doxorubicin was administered systemically at 8 mg/kg to BT474 bearingmice. Intratumor concentration of doxorubicin was analyzed by standardimmunohistochemical and fluorescence techniques previously described andstandard in the art. Doxorubicin was loaded into microwells in thedevice. The device was implanted in a BT474 tumor as in Example 7. After20 hours, the device and surrounding tissue was excised and doxorubicinconcentration in the surrounding tumor tissue was analyzed.

Results

Doxorubicin distribution is highly heterogeneous in tumors from miceadministered doxorubicin systemically. There are areas within the tumorthat have a low doxorubicin concentration (3-7 mg/kg) and areas thathave high doxorubicin concentration (8-13 mg/kg). As discussed inprevious examples, intratumor concentration of doxorubicin decreaseswith distance from the microwell when delivering doxorubicin locally bythe device. Cross-sectional analysis showing a direct comparison ofdevice and systemically dosed tumor sections revealed that the region oftissue 0-125 μm from the device on tumor sections dosed by the devicehad excessively high drug levels. In tumors dosed systemically, the125-300 μm region represents the relevant range of drug levels. FIG. 16shows a comparison of intratumor concentration of doxorubicin followingrelease from an implanted device, with a polynomial curve fit, or aftersystemic administration of doxorubicin.

Example 9 Device Measurement of Local Microdose Response is an ExcellentPredictor of Systemic Response Across Tumor Models

Materials and Methods

Murine A375, BT474, or PC3 tumor models were used to determine whetherthe device measurement of local microdose response is a satisfactorypredictor of a systemic response in different tumor models. Doxorubicinwas placed within the microwells of implantable devices. The deviceswere implanted into A375, BT474, or PC3 tumors on mice. Apoptosis, asmeasured by cleaved caspase 3 expression, was used to evaluate drugefficacy at 24 hours post implantation or systemic dosing.

To compare the efficacy of device delivered doxorubicin to efficacyachieved with systemic delivery, mice bearing A375 or PC3 tumors weresystemically administered an 8 mg/kg dose of doxorubicin. Apoptosis, asmeasured by cleaved caspase 3 expression, was used to evaluate drugefficacy at 24 hours post implantation or systemic dosing.

Results

As shown in FIG. 18, A375 tumors had the greatest apoptotic response todoxorubicin after device-delivery of doxorubicin (apoptotic index(AI)=55%) (P<0.01). BT474 tumors had an intermediate apoptotic response(AI=18%). PC3 tumors had the lowest apoptotic response (AI=6%) (P<0.01).There was little variation between samples within each tumor type. EveryA375 sample (n=12) had a greater AI than each of the BT474 samples andPC3 samples (P<0.01). Similarly, each of the BT474 samples had a greaterAI than each of the PC3 samples.

These findings correlated with results from systemic administration ofdoxorubicin. As shown in FIG. 19, local tumor apoptosis followingsystemic dosing was significantly greater in A375 tumors (AI=34.9%) thanPC3 tumors (AI=8.7%). Increased variation was observed in the systemicdosing model. This may be due to the fact that drug distribution is moreheterogeneous in systemically dosed tumors as compared to the moreprecise dosing achieved with implantable device-based delivery describedherein.

Example 10 Device Measurement of Local Microdose Response is anExcellent Predictor of Systemic Response Across Multiple Drugs and TumorModels

The implantable device assay was tested in murine tumor models (A375 andPC3) for its ability to predict the response of tumors to several othercytotoxic and targeted anti-cancer agents. Vemurafenib is an enzymeinhibitor that specifically targets the BRAF V600E mutation. A375 tumorshave this mutation. PC3 tumors do not have this mutation. Response tovemurafenib was measured by intratumor apoptotic response using theimplantable device assay. As shown in FIG. 20, response to vemurafenibwas 250% greater in the A375 model (P<0.01) as compared to the PC3model.

Gemcitabine is an inhibitor of DNA synthesis. Response todevice-delivered gemcitabine, as measured by intratumor apoptoticresponse using the implantable device assay, was greater in PC3 tumors(AI=12.5%) than the response observed in BT474 tumors (AI=2.0%). Thisresponse correlated with what is known in the art. Response todevice-delivered gemcitabine was also compared between MDA-MB231 andBT474 tumors. As shown in FIG. 21, response to gemcitabine wassignificantly greater (AI=21.8%) in MDA-MB231 tumors than BT474 tumors(AI=2.6%). This response correlated with what is known in the art.

Topotecan is a topoisomerase inhibitor and a derivative of camptothecin.Response to device-delivered topotecan was measured by intratumorapoptotic response using the implantable device assay. As shown in FIG.22, response to device-delivered topotecan was greater in PC3 tumors(AI=6.3%) than BT474 tumors (AI=2.2%) (P<0.05). This response correlatedwith what is known in the art

Example 11 Enhancement of Apoptotic Response by Addition of TargetedAgents to Doxorubicin in a Microwell of the Implantable Device

Combining cytotoxic agents with targeted agents is a promising clinicalstrategy for overcoming drug resistance in tumors. To demonstrate thecapability of the implantable device assay to test the efficacy ofcombinations of multiple compounds with great sensitivity, the effect ofthe addition of sunitinib or lapatinib to microwells in the implantabledevice already loaded with doxorubicin was evaluated. Sunitinib is amulti-kinase inhibitor. Lapatinib is a dual EGFR/HER2 inhibitor.

Materials and Methods

Sunitinib was loaded into microwells in the implantable device that werepreloaded with doxorubicin. The device was then implanted into PC3tumors. The device and surrounding tissue was removed 24 hours later andapoptosis was evaluated by cleaved caspase 3 expression using standardimmunohistochemical assays.

Lapatinib was loaded into microwells in the implantable device that werepreloaded with doxorubicin. The device was then implanted into MDA-MB231tumors. The device and surrounding tissue was removed 24 hours later andapoptosis was evaluated by cleaved caspase 3 expression using standardimmunohistochemical assays.

Sunitinib or lapatinib was loaded into microwells in the implantabledevice that were preloaded with doxorubicin. The device was thenimplanted into BT474 tumors. The device and surrounding tissue wasremoved 24 hours later and apoptosis was evaluated by cleaved caspase 3expression using standard immunohistochemical assays.

Results

In PC3 tumors, apoptosis significantly increased by 330% by the additionof sunitinib to microwells preloaded with doxorubicin. FIG. 23 shows theapoptotic response in BT474 cells after drug delivery. In BT474 tumors,apoptosis moderately increased by 66% by the addition of sunitinib tomicrowells preloaded with doxorubicin. However, lapatinib addition tomicrowells preloaded with doxorubicin increased apoptosis 355%. This wasexpected, given that BT474 is a HER2 positive tumor line. FIG. 24 showsthe apoptotic response in MDA-MB-231 cells in response to drug delivery.The apoptotic response in MDA-MB-231 cells was significantly elevated by140% (AI=17.5% to 7.4%) by the addition of lapatinib to doxorubicin inthe microwells.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. An implantable microdevice comprising: a support structurehaving microwells on a surface of or formed within the supportstructure; optionally including a compound release mechanism forcontrolling the release of an active agent from the microwells; whereinthe device is configured to permit implantation into a tissue using acatheter, cannula or biopsy needle, and wherein the device is furtherconfigured to release one or more active agents from the microwells toseparate and discrete areas of tissue adjacent to the microwell.
 2. Themicrodevice of claim 1, comprising an active agent release mechanismselected from the group consisting of the dimensions of an opening intothe microwells, a film, a membrane, a polymer matrix, and a hydrogelpad.
 3. The microdevice of claim 1 further comprising one or more activeagent or combinations of active agent within the microwells.
 4. Themicrodevice of claim 3 wherein the active agent or combinations thereofare present in different amounts.
 5. The microdevice of claim 3 havingmicrowells with different pharmacokinetic release profiles.
 6. Themicrodevice of claim 3, wherein the active agent is selected from thegroup consisting of cancer therapeutics, anti-angiogenic agent,immunomodulator, and anti-infective agents.
 7. The microdevice of claim1 further comprising a guide wire, wherein the guidewire is mechanicallycoupled to the microdevice support structure.
 8. The microdevice ofclaim 1, wherein the microwells are separated by walls or includerecessions which limit release of active agents into areas of releasefrom adjacent microwells.
 9. The microdevice of claim 1, wherein themicrodevice comprises biodegradable polymers.
 10. The microdevice ofclaim 1 wherein active agent is released from the microwells as a bolus,sustained release, delayed release, bolus followed by sustained release,and/or pulsatile release.
 11. The microdevice of claim 1 wherein theactive agent is present in solid form in the reservoir.
 12. Themicrodevice of claim 1 wherein the device does not comprise needles or afluid reservoir.
 13. The microdevice of claim 1 formed of a plasticselected from the group consisting of polyether-ether-ketone,polysulfone and polyphenylsulfone.
 14. The microdevice of claim 1 formedby methods selected from the group consisting of deep ion etching, nanoimprint lithography, micromachining, laser etching, three dimensionalprinting and stereolithography
 15. A kit comprising the microdevice ofclaim 1 and means for implantation and removal selected from the groupconsisting of a catheter, cannula and biopsy needle having an innerdiameter slightly larger than the outer diameter of the microdevice andat least one layer of cells from the tissue into which it is to beimplanted.
 16. A method for determining efficacy of a compound in vivoor in situ comprising implanting using a catheter, cannula or biopsyneed inserted into a tissue within an organism an implantablemicrodevice comprising: a support structure having microwells on asurface of or formed within the support structure, the microwells eachcontaining and releasing after implantation one or more active agentsselected from the group consisint of therapeutic, prophylactic anddiagnostic agents, the microwells optionally including a compoundrelease mechanism for controlling the release of an active agent fromthe microwells; wherein the device is configured to permit implantationinto a tissue using a catheter, cannula or biopsy needle, and whereinthe device is further configured to release one or more active agentsfrom the microwells to separate and discrete areas of tissue adjacent tothe microwell.
 17. The method of claim 16, wherein the microdevicecomprises an active agent release mechanism selected from the groupconsisting of the dimensions of an opening into the microwells, a film,a membrane, a polymer matrix, and a hydrogel pad.
 18. The method ofclaim 16 wherein the microdevice comprises two or more active agents,dosages of active agents or combinations of active agent within themicrowells.
 19. The method of claim 16 wherein the microdevice hasmicrowells releasing active agent with different pharmacokinetic releaseprofiles.
 20. The method of claim 16 wherein the microdevice isimplanted using a catheter and a guide wire, wherein the guidewire ismechanically coupled to the support structure of the microdevice. 21.The method of claim 16 wherein the microwells of the microdevice areseparated by walls or include recessions which limit release of activeagents into areas of release from adjacent microwells.
 22. The method ofclaim 16 wherein active agent is released from the microwells as abolus, sustained release, delayed release, bolus followed by sustainedrelease, and/or pulsatile release.
 23. The method of claim 16 whereinthe active agent is present in solid form in the reservoir or the devicedoes not comprise needles or a fluid reservoir.
 24. The method of claim16 wherein the microdevice is formed by methods selected from the groupconsisting of deep ion etching, nano imprint lithography,micromachining, laser etching, three dimensional printing andstereolithography.
 25. The method of claim 16 further comprisingevaluating drug efficacy in vivo or in situ by removing the microdeviceand an amount of surrounding tissue after an amount of time.
 26. Themethod of claim 25 further comprising cutting the surrounding tissuealong an axis parallel to a length of the microdevice to form a slab oftissue to be analyzed.
 27. The method of claim 25, wherein themicrodevice is removed using a coring needle.
 28. The method of claim25, wherein the thickness of the removed tissue is approximately 500 μm.29. The method of claim 25, wherein the assay is performed in vivowithout removal of the tissue adjacent to the microdevice.
 30. Themethod of claim 25, wherein the assay is performed in situ afterremoving the device and adjacent tissue from the organism.
 31. Themethod of claim 16, wherein the tissue is a tumor.